Plants generally have totipotency and, for example, can regenerate individual plants through the regeneration of adventitious shoots or adventitious embryos from undifferentiated tissues derived from somatic cells. This ability is used for, for example, the production of young-plants by cultured shoot. In addition, the technique of regenerating transformed plants via the regeneration of adventitious shoots or adventitious embryos after the introduction of genes into plant somatic cell tissues or cultured plant cells has become an indispensable technology in the field of plant biotechnology in recent years. It is generally thought that the regeneration of adventitious roots or adventitious shoots from callus, which is an undifferentiated cells or plant tissues originated from leaves, stems, and the like is regulated by the interaction of plant hormones such as auxins and cytokinins.
For plant morphogenesis, it has also been reported that a series of genes including homeobox are involved in addition to plant hormones. Homeobox genes was found as a well-conserved 183 bp DNA sequence occurring in common in certain genes that regulate the development of Drosophila. The 61 amino acid sequence translated from this region is called homeodomain, which takes a helix-turn-helix structure comprising three a helixes and which recognizes a specific nucleotide sequence thereby to bind DNA.
Animal homeobox genes have been elucidated to be transcription factors that control development processes, whereas for plants the KNOTTED1 (KN1) gene isolated from corn in 1991 is the first homeobox gene in higher plants (Vollbrecht et al., Nature 350: 241-243, 1991). Although veins of corn leaves are parallel ones, Knotted1 mutation results in disturbances in veins and drives formation of knot-like processes along veins, after which it was named Knotted.
On the other hand, using synthetic DNA corresponding to specifically highly conserved amino acid sequences in the homeobox that had been found in many animals, genomic DNA of a dicotyledon Arabidopsis thaliana was searched with a result that several homeobox genes were reported (Ruberti et al., EMBO J. 10: 1787-1791, 1991).
Homeobox genes of higher plants reported so far have been roughly grouped into five types based on the similarity of amino acid composition in the homeodomain and the structure of regions other than the homeobox domain (Tasaka, Tanpakushitu Kakusan Koso (Proteins, Nucleic Acids, Enzymes) 40(8): 1033-1042, 1995). The first type is represented by the KN1 gene of corn, the second type has the homeobox approximately in the center of a protein, of which a C-terminal end is flanked by a regularly repeated structure of leucine moieties (leucine zipper) that are involved in dimer formation of the protein. The third type has the homeodomain near the C-terminal end of a protein, and a finger structure of the metal-bound type at the N-terminal end. The fourth type, in addition to having a structure common to the third type, has repeated structures of several amino acid sequences. The fifth type has the homeobox in the N-terminal end of a protein and no other well-known characteristic structures have been found therein.
The overall homology of amino acid sequences between the different types is 32 to 58% in the homeodomain. However, as can be estimated from a report that the third helix in the homeodomain enters into the main groove of the target double stranded DNA to control transcription when a protein containing an animal homeodomain binds to DNA, this third helix has the highest homology irrespective of the type even in the gene products of plant homeoboxes. The region is thought to be essential for a homeodomain protein to bind to DNA as a transcription factor. Recently, a homeobox gene WUSCHEL was reported that does not belong to any of these five groups (Cell, 95: 805-815, 1998). Although the mutants defective in the function of the WUSCHEL gene cannot drive normal growth of apical meristem of the stem, there are no experimental reports on overexpression of the WUSCHEL gene, and it is unknown what changes may occur when the WUSCHEL gene expression is artificially enhanced.
The homeobox genes of plants have been suggested to be possibly involved in the control of organogenesis or development processes, infection protection, and regulation of material transport in the plants, details of which are not known, however. Protein having a homeobox is generally thought to serve as transcription factor, but even the target gene whose transcription is regulated by each homeodomain protein has not been elucidated. Furthermore, although the overexpression of the KN1 type among the homeobox genes causes extremely abnormal morphology, it is not known whether adventitious shoots are formed on the callus.
From the standpoint of agricultural application, a gene having a high ability of inducing adventitious shoots and branching on a cultured tissue such as callus in the tissue culture system would be considered to be useful, but there are no such genes.